Summary: We have previously shown that LLC-MK2 cells expressing the first nonstructural protein in the dengue 2 genome, NS1, can complement viruses which contain lethal mutations in the NS1 gene. We have identified a 25 amino acid region at the carboxy-terminus of the NS1 gene which is not able to be complemented by NS1. This region must be involved in an essential cis-function of the NS1 protein such as post-translational processing of the polyprotein. More detailed deletion analysis of this region has shown that 10 amino acids located -13 to -23 from the carboxy-terminus of the NS1 protein are involved in this cis-function of the NS1 protein. Further studies on these amino acids and their potential role in protein processing or viral replication are underway. In studies designed to understand the transport of NS1 in infected cells, we have demonstrated that the NS1 protein binds to the viral structural proteins, prM and E in infected mammalian cells (the cell line LLC-MK2). This was shown using pulse chase labeling experiments and by coimmunoprecipitation studies and Western blot analysis. The association between NS1 and the structural proteins was stable out to 16 hours of chase. A similar analysis of NS1 in virus containing supernatants of infected cells did not show association of NS1 with viral structural proteins. This suggests that the association between NS1 and the viral stuctural proteins in the cell cytoplasm of mammalian cells may be important for viral protein transport and targeting of viral proteins to the cell membrane where virus maturation occurs. In contrast, similar studies done in a mosquito cell line showed an association between NS1 and the viral structural proteins, prM and E in the cell cytoplasm that was not stable for more than two hours, as shown in pulse-chase experiments. Like the mammalian cells studies, NS1 was not associated with the viral structural proteins in virus containing cell supernatants. Dengue virus replicates much more efficiently in mosquito cells. Virus replication occurs more rapidly and virus titers are up to 3 logs higher than the amount of virus produced in mammalian cells. It may be that NS1 has the same activity in protein transport in mosiquito cells, but that the process is much faster and therefore the protein/protein interactions appear to be more transient. Studies are currently underway to determine the nature of the interaction betweein NS1 and the structural proteins and what this interaction means in the virus life cycle.